Yokosawa N, Tsuzuki K, Syuto B, Oguma K
J Gen Microbiol. 1986 Jul;132(7):1981-8. doi: 10.1099/00221287-132-7-1981.
Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods. The specific activity [2 X 10(4) mouse LD50 (mg protein)-1] of the toxin purified from cell extract under slightly acidic conditions was lower than that [3 X 10(5) LD50 (mg protein)-1] of the toxin purified from culture supernate under slightly alkaline conditions. Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate. The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests. The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment. In SDS-polyacrylamide gel electrophoresis, a single band of Mr 144,000 was demonstrated before trypsin treatment and two bands of Mr 100,000 and 55,000 appeared after trypsin treatment. The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment. The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps. These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract. Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.
通过两种方法从培养上清液和细胞提取物中纯化出E型肉毒梭菌毒素。在微酸性条件下从细胞提取物中纯化得到的毒素比活性[2×10⁴小鼠LD₅₀（mg蛋白）⁻¹]低于在微碱性条件下从培养上清液中纯化得到的毒素比活性[3×10⁵ LD₅₀（mg蛋白）⁻¹]。两种毒素制剂经胰蛋白酶处理后均被激活，但激活程度不同，从细胞提取物中得到的毒素激活程度比从培养上清液中得到的毒素高约30倍。如琼脂凝胶双向免疫扩散试验所示，两种毒素制剂在SDS-聚丙烯酰胺凝胶上具有相同的电泳迁移率和抗原特异性。两种毒素制剂的抗原特异性经胰蛋白酶处理后未改变。在SDS-聚丙烯酰胺凝胶电泳中，胰蛋白酶处理前显示出一条Mr为144,000的条带，处理后出现了Mr为100,000和55,000的两条条带。两种毒素制剂用¹²⁵I进行标记，并在胰蛋白酶处理前后获得了糜蛋白酶肽图。未经胰蛋白酶处理的两种毒素制剂在肽图上有许多差异，但经胰蛋白酶激活后的制剂具有相似的肽图。这些结果表明，从培养液中获得的毒素是部分激活形式，其分子构象与从细胞提取物中获得的毒素不同。胰蛋白酶处理后比活性和激活率的差异可能是由于毒素分子构象的不同。
